This application addresses broad Challenge Area (04) Clinical Research, and the specific Challenge Topic, 04-AI-101: Develop novel methods and address key questions in mucosal immunology. The long-term goal of this project is to understand how natural killer T (NKT) cells recognize and respond to microorganisms in the respiratory mucosa. We and others have previously shown that NKT cells in the lungs play a critical role in the development of airway inflammation and asthma. In addition, we have shown that glycolipids from the bacterial species Sphingomonas can directly activate pulmonary NKT cells in mice and induce airway inflammation and airway hyperreactivity (AHR), a cardinal feature of asthma. These results suggest that NKT cells in the lung mucosa may respond to other microorganisms that enter the lung, and that NKT cells could play a previously unsuspected critical role in regulating pulmonary mucosal immune responses. Although the lungs of most patients with asthma are thought to be sterile, we have strong and surprising preliminary data indicating that a highly diverse complex bacterial consortia are in fact present in the lungs of a large fraction of patients with asthma, as detected with an extremely sensitive, novel non-culture based 16S rRNA PhyloChip microarray method. The microorganisms present include Streptococcus pneumoniae, as well as bacteria not previously detected by culture-based techniques, such as Sphingomonas paucimobilis, which express glycolipids that can activate NKT cells. Based on these studies, we suggest that microorganisms are much more common in the airway mucosa of patients with chronic pulmonary inflammatory diseases than previously recognized. Furthermore, while only a few microorganisms are known to activate NKT cells, based on strong preliminary data, we hypothesize that many pulmonary microorganisms, including S. pneumoniae, Burkholderia cenocepacia, Sphingomonas paucimobilis and Aspergillus fumigatus express glycolipids that can activate NKT cells, resulting in innate and adaptive mucosal immune responses. We therefore believe that host responses to common, as well as previously unrecognized, microorganisms in the endobronchial mucosa can trigger chronic diseases in the airways. In this project, we propose to identify glycolipids from S. pneumoniae, B. cenocepacia, and A. fumigatus that can directly activate NKT cells. These studies will demonstrate a specific mechanism by which microorganisms present in the lungs can activate NKT cells and induce AHR, inflammation and asthma. Our studies will greatly expand the fundamental understanding of the types of immune responses that develop against microorganisms present in the respiratory mucosa, and how these responses result in the development of chronic lung diseases, such as asthma. PUBLIC HEALTH RELEVANCE: The results of these studies will have a direct impact on our fundamental understanding of the immune responses that occur in the respiratory mucosa. We propose to understand how a novel cell type, called natural killer T cells, fundamentally regulates innate and adaptive immunity to respiratory microorganisms. These studies will complement additional studies that we are performing, supported by other grants and using a novel, highly sensitive non-culture molecular method, to define the microorganisms that are present in the lungs of patients with chronic lung disease, such as asthma. These results will provide a much greater appreciation and understanding of mucosal immunity to microorganisms present in the respiratory tract, and how these immune responses lead to respiratory inflammation and to the development of chronic lung diseases, such as asthma and COPD.